“Human Immune System” mouse models for preclinical risk ...€¦ · Human Immune System mice Host-tumor interactions Dual tumor/HIS models for the evaluation of new anti-tumor

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“Human Immune System” mouse models for preclinical risk assessment

Nicolas LEGRAND, Ph.D. Director – Immunology

European Immunogenicity Platform 27-Feb-2019

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Research model creation services at genOway

Rodent & cell models > 2000 catalog models for target & compound specificity validation > 2000 tailor-made models Unique and broad IP platform Worldwide partnerships (academia & pharmaceutical/biotech companies)

Gene-humanized models with enhanced translatability to human •  PK and PD studies (Hu-FcRn/Hu-albumin) •  IgE mediated inflammation and allergies (Hu-IgE/Hu-FcεR1) •  inflammation & auto-immune disease (Hu-CD4, Hu-TNF-α,…) •  Immune checkpoint and co-stimulatory molecules (OX40, PD-1, GITR,…)

Cell-humanized models for prospective access to human cells in vivo •  Human hematopoietic cells (PBMC, CD34+ HSPC,…)

•  ± human tumor cells

Cytokine-producing cell reporter models

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Construction of “Human Immune System” mice

Immuno-deficient mice

Human hematopoietic cells (CD34+ HSPC; PBMC; ...)

XENOGRAFT HUMANIZATION

Human DNA GENETIC

HUMANIZATION

Mice humanized for cellular and molecular components of the hematopoietic system

Human product

EXOGENOUS HUMANIZATION

Human Immune System mice

Other tissues Tumors (PDX)

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Immuno-deficient mouse strains Shultz (2007), Nat. Rev. Immunol., 7:118

SCID NOD/SCID NSG/NOG

BRG BRGS(+)

Hematopoietic reconstitution efficiency: SCID < NOD/SCID < BRG < NSG/NOG, BRGS

(no mouse T/B/NK cells & tolerant phagocytes) mouse T/B/NK

BRG = BALB/c Rag2tm1Fwa Il2rgtm1Cgn

BRGS = BALB/c Rag2tm1Fwa Il2rgtm1Cgn SirpaNOD NSG = NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ

NOG = NOD/Shi-scid Il2rgnull

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The BRGS mouse strain

Legrand (2011), PNAS, 108:13224

‘Do-not-eat-me’

Takizawa & Manz (2007), Nat. Immunol., 8:1287

Effective CD47/SIRPα interaction to inhibit mouse

phagocyte activity towards the human xenograft

Takenada (2007), Nat. Immunol., 8:1313

‘wild-type’ allele (e.g. BALB/c)

NOD allele (NOD.Idd13)

BRGS mice

NOD Rag2-/-IL-2Rγc-/-

(BRG) BALB/c

Rag2-/-IL-2Rγc-/-

NODxBALB/c NOD.sirpa+/- Rag2-/-IL-2Rγc-/- (N1)

BALB/c Rag2-/-IL-2Rγc-/-

-> Speed congenic (BALB/c) -> SIRPαNOD allele screening (PCR)

[BALB/c Rag2tm1Fwa Il2rgtm1Cgn SirpaNOD]

•  no mouse T/B/NK cells •  tolerant mouse

phagocytes

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A simple strategy for HIS mouse generation

BRG-HIS mouse

12 weeks

Human hematopoietic reconstitution (multi-lineage; multi-organ)

HSC = Hematopoietic Stem Cells i.h. = intra-hepatic HIS = Human Immune System

Traggiai (2004), Science, 304: 104 Gimeno (2004), Blood, 104: 3886 Ishikawa (2005), Blood, 106:1565

Legrand (2008), Methods Mol. Biol., 415:65 Legrand (2011), PNAS, 108:13224

HIS mouse Immuno-deficient newborn mouse

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High human reconstitution level in BRGS-HIS mice

ü  HIS mice are routinely generated in females only;

ü  >95% of the HIS mice contain >30% hCD45+ leucocytes in blood at 12wks of age;

Blood (T+12wks) Subset distribution (blood, T+12wks)

ü  B cells 59.8 ± 7.7%

ü  T cells 10.7 ± 8.0%

ü  cDC 1.9 ± 0.6%

0 102 103 104 105

0

102

103

104

105

<PE-Cy7-A>: hCD 11c

<FIT

C-A

>: h

CD

14

33.9

16.6

48.8

hCD45+CD33+

hCD11c

hCD

14

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Restored human leucocyte homeostasis in BRGS mice

ü  BM: 2.5-fold

ü  Thymus: 2.0-fold

ü  Spleen: 7.5-fold

ü  Lymph nodes: 5-fold

Total hCD45+ (fold increase over

control BRG-HIS mice)

ü  Improved engraftment and accumulation of human hematopoietic progenitor cells (hHPC)

BM (T+12wks) Spleen (T+12wks)

ü  Enhanced accumulation of all human hematopoietic subsets, in particular T & NK cells

Legrand (2011), PNAS, 108:13224

BRG BRGS

ü  Selective improvement of T cell homeostasis

CD47/SIRPαcompatibility

no (BRG)

yes (BRGS)

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BRGSF-HIS mice for enhanced human DC density DC niche

Tilting the balance towards higher density of human DC in HIS mice

ü  the cytokine FLT3L/FLK2L is crucial for steady-state dendritic cell development;

ü  mouse and human FLT3L are highly homologous (cross-reactive);

ü  FLK2-/- mice show reduced numbers of mouse DC;

BRGSF mice

BRGSF = BALB/c Rag2tm1Fwa Il2rgtm1Cgn SirpaNOD Flk2tm1Irl Li (2016), EJI, 46:1291

BRGSF-HIS FLT3-L

Blood D0 D2 D4 D7 D8

ü  “human DC boost on demand” hFLT3L injection into HIS mice increases human DC numbers

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Human leukocyte reconstitution – optimization strategies

BRG-HIS

hHSC hB hT hDC hNK mDC hM/G

<<

BRGS-HIS <<

BRGSF-HIS +FLT3L

>>

BRGSF-HIS ±

Identification of limiting factors for human leukocyte homeostasis in BRG-HIS mice:

ü  Human cytokine bio-availability:

IL-7 (thymopoiesis; αβ T cells)

IL-15/IL-15Rα (thymopoiesis; memory αβ T4/T8; T γδ; NK)

TPO (hHSC)

IL-3/GM-CSF; M-CSF (myeloid cells)

FLT3-L (cDC; pDC; mono/granulocytes; NK)

ü  Human dendritic cell density (αβ T cells)

ü  Human MHC expression (αβ T cells)

ü  Compatible CD47/SIRPα signaling axis (mouse phagocyte tolerance)

all human cell subsets benefit from this single modification

Legrand (2009), Cell Host & Microbe, 6:5 Huntington (2009) JEM, 206:25

van Lent (2009), J. Immunol., 183:7645 Chen (2009), PNAS, 106:21783 Strowig (2009), JEM, 206:1423

Shultz (2010), PNAS, 107:13022 O’Connell (2010), PLOS ONE, 5:e12009

Rongvaux (2011), PNAS, 108:2378 Willinger (2011), PNAS, 108:2390

Huntington (2011), PNAS, 108:6217 Legrand (2011), PNAS, 108:13224 Strowig (2011), PNAS, 108:13218

Huntington (2011), EJI, 41:2883 Suzuki (2012), Int. Immunol, 24:243

Li (2013), J. Immunol., 191:3192 Ding (2014), J. Immunol., 192:1982 Li (2016), Eur. J. Immunol., 46:1291

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A flexible tool for research & industrial applications

CRO services for outsourced R&D

Human Immune System mice

Host-tumor interactions Dual tumor/HIS models for the evaluation of new anti-tumor compounds relying on the activity of human

immune cells (cell lines & personalized PDX);

‘Ready-to-use’ immuno-deficient mice

Delivery to end-users

Experimental human hematopoiesis

human hematopoietic cell maintenance, differentiation

and function

Pre-clinical POC candidate vaccines; bio-

therapeutics & mAbs; gene therapy; immunogenicity;

adjuvants; immuno-modulatory molecules;

inflammation;

Infectious diseases Screening of new anti-viral compounds, microbicides,

antibiotics; prospective experimentation for various

human leucocyte-tropic pathogens

HIV, DNV, HTLV, EBV, HCMV, Yellow fever, Measles

S. Typhi, M. Tuberculosis,…

‘Ready-to-use’ HIS mice Delivery to end-users

(‘humanization factory’)

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Human immune responses in HIS mice

Traggiai (2004), Science, 304:104

ü  commercial TT vaccine, 1/10 of the human dose i.p. per injection (1 immunization + 2 boosts)

ü  analysis 10 days after last boost (5-6 wks after onset of vaccination)

Baenziger (2006), PNAS, 103: 15951

ü  poor anti-HIV B cell response (IgG in 1/25 YU-2/R5 infected animals)

ü  no/weak anti-HIV T cell response

ü  similar results with other model pathogens e.g. measles; influenza; yellow fever

Analysis of B cell responses at clonal level

ü  weak responses ~0.2-1% specific IgG levels of vaccinated humans

ü  low efficiency of i.p. route 5-10% responder HIS mice

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From primary B cells to human monoclonal antibodies

IL-21

L cells

3

B

B

B

B cells (CD27+)

1

pLZRS (retroviral construct)

2

- Differentiation

+ Survival

Tracking marker

CD40 CD40L

B B B B B B B cell clones

B B B

5 70

CD19

GFP

4

Single-cell sorting

Human B cells

Kwakkenbos & Diehl (2009), Nat.Med., 16:123

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Vaccination scheme

7-10 days

i.m. i.m. >20% huCD45+

in blood

Becker & Legrand (2010), PLoS ONE, 5:e13137

Control (PBS) Vaccine (Tetanus; HBV) 1/10 human dose

BRG-HIS mouse (4-month old)

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Immunological parameters after vaccination Plasma (Ag-specific Ig)

Spleen

Plasma (total Ig)

~40% responders

(+)

Becker & Legrand (2010), PLoS ONE, 5:e13137

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Antigen-specific B cell clones generated from HIS mice

CD27+ B cells

B cell clones: 15 HBsAg-specific, IgM+ 18 TT-specific, IgM+ (frequency ~1:250)

O.D

. (Ig

M)

Origin of tested antigens

ELISA (TT clones)

Becker & Legrand (2010), PLoS ONE, 5:e13137

Sec

rete

d H

B-A

g (%

con

trol i

nfec

tion)

0

5 0

1 0 0

1 5 0

2 0 0

HBsAg HBeAg

HBV mAb:HBV

+ -

+ 1:9

+ 1:1

Control mAb MA18/7

+ 1:9

+ 1:1

HIS mAb-1 2β2-D6-11

+ 1:9

+ 1:1

HIS mAb-2 3β2-G1-7

- -

Neutralization assay (HBsAg clones)

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All clones show low levels of hypersomatic mutations IgM VH amino-acid sequence (HBsAg-specific B cell clones)

Becker & Legrand (2010), PLoS ONE, 5:e13137

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Human immune responses in HIS mice Human B cell responses are induced in HIS mice upon vaccination ü  Human B cell responses are induced by various vaccines (OVA; KLH; TT; HBV; Flu)

ü  Immunization per i.m. route is more efficient than i.p. route

ü  Human antigen-specific B cells can be isolated and cloned from vaccinated HIS mice ü  Optimized HIS mouse models with improved human cell homeostasis & function permit isolation of large numbers of antigen-specific B cell clones (including IgG+) e.g. in humanized BRGS mice

ü  The generated mAbs exhibit features of low affinity, close-to-germline antibodies (limited germinal center reaction; limited Ig switch; limited affinity maturation process)

Optimizating immunization strategies ü  Vaccination design (protocols, timing, formulation, adjuvants) ü  Incremental, step-wise optimization of new recipient mouse strains for improved xenograft features (human cell content and function)

ü  Use of exogenous agonists of immune cells during immunization

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Vaccination in hDC-boosted BRGSF-HIS mice

Di Santo & Mention (2010), patent Chen (2012), J. Immunol., 189:5223 Ding (2014), J. Immunol., 192:1982 Li (2016), Eur. J. Immunol. 46:1291

FLT3-L more hDC GM-CSF/IL-4 more mature hDC

ü  Human antigen-presenting cells might represent a functional bottleneck – human DC boosting strategy?

ü  Model antigen: Keyhole Limpet Hemocyanin (~3400 a.a.; ~390kDa)

KLH (K) vs. KLH/Alum (K/A)

ü  Exogenous agonists of immune cells before immunizations: soluble FLT3-L/Fc + hydrodynamic delivery of hGM-CSF/hIL-4 encoding DNA plasmids

Birth

FLT3-L GM-CSF/IL-4

hHPC injection

wk <1

Evaluation of humanization

wk 11

wk 12-13

Prime Boost-1

wk 14

wk 17

wk 22

Full body analysis

FLT3-L

Boost-2

wk 20

FLT3-L

ü  Standardized vaccination design

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Human B cell responses to KLH

ü  Human IgM responses 83% (20/24) responding animals in KLH-specific IgM ELISA vs. 20-40% w/o immuno-stimulation

ü  Human IgG responses 45% (9/20) of the IgM-responder animals also exhibit anti-KLH IgG responses vs. almost no detectable IgG w/o immuno-stimulation

ü  Impact of adjuvant No benefit from alum

CT: PBS-injected K: KLH-injected

K/A: KLH/alum-injected

ELISA for anti-KLH IgM/G

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Human T cell responses to KLH

ELISPOT IFNγ (spleen)

- PHA KLH

Control BRGSF-HIS

Vaccinated BRGSF-HIS

KLH + αHLA-II

ü  Human T cell responses 67% (14/21) of the animals show a T cell response to KLH vs. no detectable T cell responses without immuno-stimulation

ü  MHC restriction of T cell responses 57% (8/14) of the responder animals show partial HLA class-II restriction (human DC biased?) HLA-class-I and mouse MHC restricted responses possible as well

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An unprecedented level of human immune responses

Enhancement of human dendritic cell density and maturation status has major functional benefits on human immune responses in HIS mice

hT hDC mDC

BRGS

hT/hDC encounter

Low probability

‘Boosted’ BRGSF

High probability

ü  Screening of candidate vaccines ü  Validation of immuno-modulatory compounds ü  Evaluation of new adjuvants ü  Evaluation of compound immunogenicity ü  Model limitations should be kept in mind…

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Human immune responses to tumor cells in HIS mice

ü  ‘Double-humanized’ mice - Human immune system - Human tumor xenograft

ü  Daudi-luc+ cell model B cell leukemia (BCL) [i.v.]

Human tumor cells

ü  Tumor cell outgrowth Tumor cells can be tracked by bioluminescence

ü  Human immune cells limit tumor outgrowth

BRGS-HIS BRGS (non-HIS)

(42d) (84d)

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BCL/HIS mice for bispecific antibody POC study

Human B cell depletion in blood * p<0.05; ** p<0.01 (compared to HIS-CT)

ü  Transient B cell depletion activity is observed with the 2 B cell-targeting test products (mAb2 & mAb3)

ü  Evaluation of treatment anti-tumor efficacy 14-weeks old BRGS-HIS mice 5x106 Daudi-Luc+ cells i.v. Comparison of 3 Bs-mAbs 2 injections (d3/7; 1mg/kg i.v.)

CT: PBS vehicle control mAb1: human T cell targeting only (monovalent) mAb2: human T cell + human B cell targeting (bispecific) mAb3: human B cell targeting only (monovalent)

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BCL/HIS mice for bispecific antibody POC study ü  Evaluation of treatment anti-tumor efficacy

14-weeks old BRGS-HIS mice 5x106 Daudi-Luc+ cells i.v. Comparison of 3 Bs-mAbs 2 injections (d3/7; 1mg/kg i.v.)

ü  Monovalent B cell targeting does not efficiently control tumor outgrowth (mAb3)

ü  Tumor control is only achieved when human T cells are recruited to the tumor B-cells by the bispecific antibody (mAb2)

Tumor cell expansion

CT: PBS vehicle control mAb1: human T cell targeting only (monovalent) mAb2: human T cell + human B cell targeting (bispecific) mAb3: human B cell targeting only (monovalent)

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Conclusions Human immune responses in HIS mice ü  Human dendritic cells (number & maturation status) represent a limitation for the consistent generation of human antigen-specific immune responses in HIS mice

ü  Human B cell responses are induced upon vaccination, but remain weak/rare without immuno-stimulation

ü  Human antigen-specific B cells can be isolated and cloned from vaccinated HIS mice, but the generated mAbs exhibit features of low affinity, close-to-germline antibodies

ü  Human T cell responses are observed in a large majority of human DC-boosted animals

ü  A major field bottleneck has been removed with such an immuno-stimulatory approach

ü  Human immune cells efficiently respond to tumors (allogeneic tissues) and efficiently respond to anti-tumor, mAb-induced effector cell mobilization

Major model improvements over the past 30 years… and still ongoing ü  Standardized HIS mouse production ü  Optimized immuno-deficient mouse models with combined genetic/cellular humanization strategies

ü  Addressing the specific requirements/limitations of each application field e.g. for immunogenicity assessment: expected frequency of response vs. batch size? Immune tolerance for tested products? Genetic variability of human HSC donors? Optimization of vaccination strategies?

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“Human Immune System” mouse models for preclinical risk assessment

Nicolas LEGRAND, Ph.D. Director – Immunology

European Immunogenicity Platform 27-Feb-2019